Method for determining the amount of template nucleic acid present in a sample

ABSTRACT

A method for determining the amount of template nucleic acid present in a sample comprising the steps of: I) bringing into association with the sample all the components necessary for nucleic acid amplification, and all the components necessary for a bioluminescence assay for nucleic acid amplification and subsequently: ii) performing the nucleic acid amplification, iii) monitoring the intensity of light output from the bioluminescence assay; and iv) determining the amount of template nucleic acid present in the sample.

This application is the U.S. national phase of International PatentAppln. No. PCT/GB2004/000127, filed 14 Jan. 2004, which designated theU.S. and claims priority benefit of GB 0300802.6, filed Jan. 14, 2003;the entire contents of each of which are hereby incorporated byreference.

The invention relates to a method for determining the amount of templatenucleic acid present in a sample comprising the steps of: i) bringinginto association with the sample all the components necessary fornucleic acid amplification, and all the components necessary for abioluminescence assay for nucleic acid amplification and subsequently:ii) performing the nucleic acid amplification reaction; iii) monitoringthe intensity of light output from the bioluminescence assay; and iv)determining the amount of template nucleic acid present in the sample.

BACKGROUND

Nucleic acid amplification may be used to determine whether a particulartemplate nucleic acid is present in a sample. If an amplificationproduct is produced, this indicates that the template nucleic acid waspresent in the sample. Conversely, the absence of any amplificationproduct indicates the absence of template nucleic acid in the sample.Such techniques are of great importance in diagnostic applications, forexample, for determining whether a pathogen is present in a sample.

Nucleic acids may be amplified by a variety of thermocycling andisothermal techniques. Thermocycling techniques, such as the polymerasechain reaction (PCR), use temperature cycling to drive repeated cyclesof DNA synthesis leading to large amounts of new DNA being synthesisedin proportion to the original amount of template DNA. Recently, a numberof isothermal techniques have also been developed that do not rely onthermocycling to drive the amplification reaction. Isothermal techniqueswhich utilise DNA polymerases with strand-displacement activity havebeen developed for amplification reactions that do not involve anRNA-synthesis step. Similarly, for amplification reactions that doinvolve an RNA-synthesis step, isothermal techniques have been developedthat use reverse transcriptase, RNase H and a DNA-dependent RNApolymerase.

The products of nucleic acid amplification reactions have traditionallybeen analysed using gel electrophoresis (either agarose oracrylamide-based) using a fluorescent dye (such as ethidium bromide) tostain for the presence of DNA. This method can be used to indicate thenumber, amount and size of the amplified products. However, thepreparation, running and analysis of amplification reactions using gelelectrophoresis requires extensive manual intervention and hazardousreagents and is time-consuming (typically taking around 1 hour intotal). In addition, multiple PCR cycles (typically 30) are required toproduce detectable product. More recently, methods with increasedsensitivity over gel electrophoresis have been developed which rely onfluorescence-based techniques or a turbidity assay to monitor theproducts of nucleic acid amplification reactions in real-time.

A characteristic of DNA and RNA polymerases is the fact that theyrelease the compound pyrophosphate (PPi) each time they incorporate anew base into the growing DNA/RNA molecule. Thus PPi is produced as aside product in a stoichiometric amount as nucleotides are added to agrowing nucleotide chain by the polymerase. Thus it follows that theconcentration of PPi is proportional to the amount of nucleic acidsynthesis that has occurred and therefore to the accumulation ofamplicon. For a polymer of length n, the reaction may be shown as:

A sensitive assay for PPi is known as the Enzymatic LuminometricInorganic Pyrophosphate Detection Assay (ELIDA) (see Nyren, P. andLundin, A., Anal. Biochem. 151: (2) 504-509 (1985)). This assay has twosteps: (1) conversion of pyrophosphate (PPi) to ATP by the enzyme ATPsulphurylase, and (2) utilisation of the ATP to produce light in thepresence of luciferin and oxygen, catalysed by luciferase:

The use of ELIDA-type assays is advantageous in that bioluminescencereadings can be rapidly obtained from small sample volumes and thereadings can be made using simple, cheap monitoring devices such asphotographic film or charge-coupled device (CCD) cameras.

U.S. Pat. No. 5,534,424, U.S. Pat. No. 5,498,523, WO 98/28440, WO98/13523 and WO 02/20836 describe the use of ELIDA-based methods forsequencing short regions of DNA. The ELIDA assay was used to follow theincorporation of single nucleotides into a DNA molecule by a polymeraseduring a single round of polymerisation during pyrosequencing.Pyrosequencing is an iterative technique whereby only one of the fourdeoxynucleotide triphosphates (“dNTPs”) is present in each of theiterative assays to enable each deoxynucleotide triphosphate (“dNTP”) tobe tested at each position of the sequence. Thus all of the componentsnecessary for DNA synthesis are never present simultaneously.

The use of an end-point ELIDA-type assay termed ‘H3PIM’ for monitoring athermocycling polymerase chain reaction (“PCR”) has also been described(see WO 92/16654 and Tarbary et al., J. Immunological Methods, 156(1992) 55-60). Aliquots of the reaction mixture were taken atpredetermined regular time intervals throughout the reaction processand/or at the end of the amplification process. Thus a lengthy stepwiseassay involving the multiple addition of reagents is described.

WO 02/064830 describes the use of an ELIDA assay to perform an end-pointassay for monitoring a thermocycling PCR reaction. In WO 02/064830 theELIDA assay can be performed in a single step, whereas in WO 92/16654multiple additions and an incubation step are required for monitoringthermocycling PCR as an end-point assay.

There are a number of problems associated with the end-point assaysdescribed above. Firstly, they require the components of thebioluminescence assay to be added to the reaction mixture following theamplification reaction. Opening of the tube may lead to contamination ofthe sample and moreover, to contamination of the laboratory. If thesample itself becomes contaminated then this could result infalse-positives or false-negatives being generated. Moreover, if thelaboratory becomes contaminated with the amplified template nucleicacid, this increases the likelihood that future samples will becomecontaminated and false-positive results or false-negative results beingobtained (for example, see Victor, T. et al., ‘Laboratory experience andguidelines for avoiding false-positive polymerase chain-reactionsresults’, Eur. J. Clin. Chem. & Clin. Biochem., 31(8): 531-535 (1993)).Thus the possibility of contamination represents a severe disadvantagein the use of end-point analysis of this type in diagnostic methods.

A further problem with the use of end-point analysis as described aboveis that dATP also acts as a substrate for luciferase. Thus when dATP isused as a substrate for the polymerase, spectral interference resultsfrom dATP instead of ATP reacting with the luciferase. WO 02/064830describes how when dATP is used as the substrate in the amplificationreaction, the light signal from the ELIDA rapidly decays. This decaywould be a serious obstacle to the utility of an endpoint assay as thelight reading measured would not only be a function of PPi concentrationbut also of time. Hence, if the endpoint assays are not performed withstrict timing, they will not be quantitative.

An alternative to end-point assays are assays which are able to monitorthe synthesis of nucleic acid during an amplification reaction in‘real-time’, i.e., as the nucleic acid synthesis is progressing.Existing real-time assays include fluorescence-based techniques andturbidity assays.

Fluorescence-based techniques work by monitoring the change influorescence that is associated with the accumulation of anamplification product by some means. For example, methods for monitoringthe amplification of DNA during PCR using double-stranded DNA-bindingdyes (specifically hybridisation probes containing donor and acceptorfluorophores) are described in U.S. Pat. No. 5,994,056, WO 97/44486, WO99/42611 and U.S. Pat. No. 6,174,670. These real-time fluorescence-basedtechniques make it possible to follow PCR without liquid sampling, thusavoiding the need for the reaction tube to be opened and thereforedecreasing the risks of contamination.

However, fluorescence-based techniques have significant drawbacks. Inparticular, the cost of fluorescent reagents, particularlyfluorescently-labelled primers, is high and sample preparation can becumbersome. Further, the application of fluorescence-based systems maybe hampered by the limited capacity of equipment and its high cost.Normally, a computer-driven integrated thermocycler-fluorimeter isrequired as the methods often follow PCR in real-time rather than beingemployed for end-point analyses. As a result, the accessibility (cost),and portability of such systems is compromised. Since detection iscarried out within the PCR instrument, such methods are only availableto suitably equipped laboratories.

Real-time turbidity assays involve monitoring the presence or absence ofa white precipitate of magnesium pyrophosphate in the amplificationreaction mixture as a method of determining whether PPi has beenproduced. This has been described as a method for determining whether ornot an isothermal loop-mediated amplification reaction has occurred (seeMori, Y. et al., ‘Detection of loop-mediated isothermal amplificationreaction by turbidity derived from magnesium pyrophosphate formation’,Biochem. and Biophys. Res. Comm., 289, 150-154 (2001)). However, thismethod is not very sensitive and requires PPi concentrations of around0.6 mM before significant turbidity is observed.

SUMMARY OF THE INVENTION

The invention provides a method for determining the amount of templatenucleic acid present in a sample comprising the steps of:

-   -   i) bringing into association with the sample all the components        necessary for nucleic acid amplification, and all the components        necessary for a bioluminescence assay for nucleic acid        amplification including:        -   a) a nucleic acid polymerase,        -   b) the substrates for the nucleic acid polymerase,        -   c) at least two primers,        -   d) a thermostable luciferase,        -   e) luciferin,        -   f) optionally ATP sulphurylase, and        -   g) optionally adenosine 5′ phosphosulphate,            and subsequently:    -   ii) performing the nucleic acid amplification reaction;    -   iii) monitoring the intensity of light output from the        bioluminescence assay, and    -   iv) determining the amount of template nucleic acid present in        the sample.

PPi is produced as a consequence of nucleic acid polymerisation duringthe amplification reaction. A method of the invention involves couplingthis production of PPi to light output from the bioluminescence assay.Preferably, the PPi is first converted to ATP. The ATP is then detectedby a bioluminescence assay catalysed by a luciferase that uses ATP as asubstrate for the production of light in the presence of luciferin andoxygen. Thus the luciferase is used to follow changes in theconcentration of ATP. Preferably, this is achieved using an ELIDA-typeassay in which PPi is converted to ATP by ATP sulphurylase and then theATP is used by the luciferase to produce light. Alternatively, PPi isdetected directly by the luciferase. By monitoring the intensity oflight output from the bioluminescence assay, it is possible to determinehow much PPi is present in the reaction mixture and thereby determinethe amount of template nucleic acid present in the sample. Thus themethod assays the in vitro enzymatic synthesis of nucleic acid and makesit possible to quantify the extent to which the nucleic acid has beenamplified as a result of de novo polymerisation during the amplificationreaction.

The nucleic acid amplification reaction of step ii) can be equated witha “processive” nucleic acid polymerase reaction in that more than onenucleotide addition cycle is carried out without further additions to ormanipulation of the buffer components.

The presence of the luciferase and other components of thebioluminescence assay during the amplification reaction of step ii)greatly simplifies the analysis of the sample as it obviates therequirement for further manipulation of the reaction mixture once theamplification reaction has begun. For example, it is not necessary totake aliquots of the sample in order to determine how much PPi has beenproduced. Instead, the bioluminescence assay is performed directly onthe reaction mixture used for the enzymatic nucleic acid amplificationreaction in the presence of all the components necessary for the nucleicacid amplification reaction, i.e., on the reaction mixture that isformed in step i). Neither is it necessary to add the components of thebioluminescence assay to the reaction mixture during or following theamplification reaction.

The components of the bioluminescence assay (also known as the‘pyrophosphate assay’ or ‘PPi assay’) and the amplification reactionmust be able to withstand the conditions of the nucleic acidamplification reaction of step ii). For example, a thermostable ATPsulphurylase and/or thermostable luciferase and/or thermostable nucleicacid polymerase can be used. The term ‘thermostable’ as used herein inrelation to an enzyme, refers to an enzyme that is stable within thetemperature range at which the nucleic acid amplification reaction ofstep ii) is carried out.

The components of step i) are preferably stabilised by lyophilisation orby the presence of stabilising factors. Thus stabilisers are alsopreferably brought into association with the sample in step i). Forexample one or more of BSA, trehalose, polyvinylpyrrolidone anddithiothreitol (DTT) may be brought into association with the sample instep i). Preferably, all of these stabilisers are brought intoassociation with the sample in step i).

The temperature and time required for nucleic acid amplificationreactions are considerably different from those required for nucleicacid polymerisation reactions. Nucleic acid amplification reactionsrequire either a high temperature or a long duration (e.g. 15 minutes to24 hours) or both. In contrast, nucleic acid polymerisation reactionscan be rapidly carried out at low temperatures (e.g. 37° C.).Luciferases are known to be unstable. For example, wild-type fireflyluciferase rapidly inactivates at 37° C. Luciferases are also known tobe easily inhibited, for example by oxyluciferin, the product of its ownlight reaction. However, it has surprisingly been found that luciferasescan remain stable during the nucleic acid amplification reaction of stepii). Furthermore, it has been found that luciferases can remain stableduring the entire course of the nucleic acid amplification reaction ofstep ii). This is surprising due to the long duration required forcertain nucleic acid amplification reactions.

The thermostable luciferase that is brought into association with thesample in step i) is a luciferase enzyme that is stable within thetemperature range at which the nucleic acid amplification reaction ofstep ii) is carried out. The particular luciferase used will depend uponthe conditions under which the nucleic acid amplification reaction ofstep ii) is performed. The term ‘luciferase’ as used herein refers to anenzyme that catalyses a bioluminescent reaction. Luciferases that aresuitable for use in the methods of the invention include both wild-typeluciferases and mutant or variant luciferases, provided that these arestable within the temperature range at which the nucleic acid reactionof step ii) is carried out. An example of a thermostable luciferase thatis suitable for use in a method of the present invention is theUltra-Glow thermostable luciferase from Promega.

The nucleic acid amplification reaction of step ii) may or may notinvolve a RNA synthesis step. In methods in which the amplificationreaction of step ii) does not involve an RNA synthesis step, thesubstrates for the polymerase include each of the four dNTPs: dATP,dTTP, dCTP and dGTP. One or more of the dNTPs may be replaced with asuitable analogue thereof. In these embodiments, the luciferasepreferably uses ATP as a substrate for the production of light. Examplesof luciferases which use ATP as a substrate for the production of lightare firefly luciferase (from Photinus pyralis) and mutants thereof.Preferably, the luciferase which uses ATP as a substrate for theproduction of light is the Ultra-Glow thermostable luciferase fromPromega. In embodiments in which the luciferase is used to followchanges in the concentration of ATP, ATP sulphurlyase is present in thereaction mixture. Preferably, the embodiments in which the luciferase isused to follow changes in the concentration of ATP are those embodimentsin which the amplification reaction of step ii) does not involve an RNAsynthesis step. Alternatively, a luciferase may be used that itselfbehaves like an ATP sulphurylase in addition to catalysing thebioluminescence assay. In such cases, it is not necessary to add ATPsulphurylase to the reaction mixture in step i).

Adenosine 5′ phosphosulphate is required for ATP sulphurylase to produceATP from PPi and is added to the reaction mixture in step i) when ATPsulphurylase is present and also when a luciferase is used that itselfbehaves like an ATP sulphurylase in addition to catalysing thebioluminescence assay.

For amplification reactions that do involve an RNA synthesis step, thesubstrates for the polymerase include each of the four dNTPs (dATP,dTTP, dCTP and dGTP) and each of the four nucleotide triphosphates(“NTPs”) (ATP, UTP, CTP and GTP). One or more of the dNTPs and/or NTPsmay be substituted by a suitable analogue. Thus when the amplificationreaction involves an RNA synthesis step, endogenous ATP is present inthe reaction mixture as one of the substrates for the polymerase unlessan ATP analogue is used that can be used by the RNA polymerase but doesnot react with luciferase. Significant amounts of endogenous ATP in thereaction mixture would severely compromise the use of a method of theinvention in which the luciferase brought into association with thesample in step i) is required to be sensitive to small changes in theconcentration of ATP. In order to overcome this problem, areversibly-inhibited luciferase is preferably used in embodiments inwhich the nucleic acid amplification reaction of step ii) involves anRNA-synthesis step and endogenous ATP is present in the reactionmixture. The term ‘reversibly-inhibited luciferase’ as used hereinrefers to a luciferase which has become inhibited by a component otherthan PPi, but which inhibition is relieved by low concentrations of PPi.For example, luciferases are known to become inhibited by oxyluciferin,the product of their own reaction. This inhibition has been found to berelieved by low concentrations of PPi. Thus the use of areversibly-inhibited luciferase enables PPi to be detected directly bythe luciferase since PPi has direct effects on an inhibited luciferase.A series of control reactions using different concentrations of thetemplate nucleic acid can be carried out to determine the time taken forthe inhibition of the reversibly-inhibited luciferase to be relieved byPPi for particular concentrations of the template nucleic acid.

The reversibly-inhibited luciferase may be inhibited by a componentother than PPi prior to adding the luciferase to the reaction mixture instep i). Alternatively, the reversibly-inhibited luciferase may beformed in situ due to the presence of the inhibitor in the reactionmixture. Preferably, the reversibly-inhibited luciferase is a luciferasethat in its uninhibited state uses ATP to produce light. In particular,the luciferase is preferably a beetle luciferase and is preferably afirefly luciferase.

In embodiments in which the luciferase brought into association with thesample in step i) of a method of the invention is a reversibly-inhibitedluciferase, ATP sulphurylase and adenosine 5′ phosphosulphate are notbrought into association with the sample in step i). However, inembodiments in which the nucleic acid amplification reaction of step ii)involves an RNA synthesis step and a suitable ATP analogue that is asubstrate for the RNA polymerase but not for luciferase (or at least, isa very poor substrate for luciferase) is brought into association withthe sample in step i) rather than ATP itself, then the luciferase thatis brought into association with the sample in step i) can be aluciferase which uses ATP for the production of light and then ATPsulphurlyase and preferably adenosine 5′ phosphosulphate will then bebrought into association with the sample in step i).

A reversibly-inhibited luciferase may also be used in embodiments of theinvention in which the nucleic acid amplification reaction of step ii)does not involve an RNA-synthesis step. In such cases, ATP sulphurylaseand adenosine 5′ phosphosulphate will not be brought into associationwith the sample in step i).

A further advantage of a method of the invention is the ease with whichthe light output in step iii) can be detected. Preferably, the intensityof light output in step iii) is monitored visually. Suitable methods formonitoring the intensity of light output include using photographic filmor a charge-coupled device (CCD) camera. Alternatively, the intensitymonitoring the intensity of light output from the bioluminescence assayusing a CCD camera. The light output may be amplified for visualisationwhere necessary. Thus the ability to detect the light output using onlyphotographic film or a CCD camera has the advantage over techniqueswhich employ fluorescence analysis or gel-based analysis in that nocomplex hardware or optics are required. Furthermore, the intensity oflight output can be monitored without the need to irradiate the samplein any way (as is required in techniques involving fluorescence orabsorbance), without the need for any electrochemical interface with thesample (e.g. as in semi-conductor-based approaches: Wilding, P. et al.,(1994) ‘PCR in a silicon microstructure’, Clinical Chemistry, 40(9):1815-1818) or without the need for indirect irradiation (e.g. as inSurface Plasmon Resonance approaches: Bianchi, N. et al., (1997)‘Biosensor technology and surface plasmon resonance for real-timedetection of HIV-1 genomic sequences amplified by polymerase chainreaction’, Clinical and Diagnostic Virology, 8(3): 199-208).

Further, one or more than one (for example thousands) of samples, may bemonitored simultaneously, for example by a single CCD camera. Thus, amethod of the invention may use simple, cheap hardware, with thepossibility of portability and miniaturisation and easy integration intohigh throughput systems.

Step i) of a method of the invention also preferably includes bringing asuitable buffer into association with the sample. Buffers which aresuitable for use with a method of the invention include buffers whichenable the amplification reaction to proceed and also which enable thebioluminescence assay to proceed. Preferably, the buffer comprises asource of magnesium ions. These are preferably in the form of MgCl₂ orMgSO₄. For example, a suitable buffer may contain Tris-acetate,potassium chloride, ammonium sulphate, magnesium sulphate and tritonX-100 at pH 8.8 at 25° C.

Advantageously, at least steps ii) and iii) of a method according to theinvention are carried out in a sealed vessel. This is of great utilitysince the ability to perform both the amplification reaction and thebioluminescence assay in a sealed vessel reduces or even prevents thepossibility of the sample becoming contaminated. Moreover, it reduces oreven prevents the possibility of the laboratory becoming contaminated.This is particularly important as if even one copy of the templatenucleic acid were to escape into the laboratory, this could potentiallycontaminate other samples to be tested and give false-positive results.Thus, the ability to prevent contamination is of particular importancewhere a method of the invention is used in a diagnostic application.

In order to further prevent contamination, following step iv) the vesselis preferably subjected to a suitable treatment in order to destroy thenucleic acid contained in it, in particular to destroy the templatenucleic acid. The vessel is itself also preferably destroyed followingstep iv) or following destruction of the nucleic acid contained in it.This minimises the possibility of the lab and/or further samplesbecoming contaminated.

Preferably, in step iii) of a method of the invention, the intensity oflight output is monitored during the nucleic acid amplificationreaction. This is only possible as a result of the components for thebioluminescence assay being present throughout the nucleic acidamplification reaction of step ii). Preferably, the intensity of lightoutput is monitored over the time course of the nucleic acidamplification reaction, i.e., from the beginning to the end of thenucleic acid amplification reaction. Alternatively, the intensity oflight output may be monitored during at least a part of the nucleic acidamplification reaction. Alternatively and/or additionally, intensity oflight output can be monitored after the nucleic acid amplificationreaction of step ii) has finished and/or prior to the amplificationreaction of step ii) beginning, for example, in order to take a controlreading. The ability to monitor the intensity of light output during theamplification reaction of step ii) simplifies the handling of thereaction vessel and also enables a rapid determination of the amount oftemplate nucleic acid present in the sample. A further advantage ofmonitoring the intensity of light output during the course of theamplification reaction is that any background signal that is produced bydATP reacting with the luciferase does not interfere with the method ofthe invention. This only becomes an issue with end-point analysis.

Preferably, step iii) of a method of the invention further includesproducing a data set of intensity of light output as a function of time.The data set is used to determine the amount of template nucleic acidpresent in the sample. Preferably, the data set is analysed by asoftware application and/or is represented in the form of a graph or alist of figures. For example, the data set may be represented as a plotof light intensity over time or a plot of the rate of change in lightintensity over time (i.e., the first derivative).

The intensity of light output may be monitored at one or morepredetermined times. These predetermined times are preferably atpredetermined times following the time at which all the conditionsnecessary for the nucleic acid amplification reaction of step ii) totake place are present, at which time (t)=0 mins. Such conditions arethat a reaction mixture has been formed as set out in step i) and thatthe reaction mixture is at a suitable temperature for amplification toproceed, said temperature also being a temperature at which thecomponents of the amplification reaction and the bioluminescence assayare stable. For example, the intensity of light output may be monitoredat set predetermined time intervals during at least a part of theamplification reaction. Preferably, the intensity of light output ismonitored at set predetermined time intervals during the wholeamplification reaction. For example, these intervals could be every 30seconds, every 1 minute, every 1 minute 30 seconds, etc. Alternatively,the intervals between predetermined times may vary. Preferably, one, twoor more light readings are taken per minute. The more readings that aretaken per minute, the greater the confidence in the results will be andthus it is preferable to take as many readings per minute as possible.Preferably, the light output is first monitored at time=0 mins. Incertain embodiments, the intensity of light output may also be monitoredafter the amplification reaction has finished.

The greater the sensitivity of the light detection system being used,the more time points per minute are possible since when using a moresensitive camera, each datum comes from integrating the light emissionover a shorter time than with a less sensitive CCD camera. Thus it isadvantageous to use as sensitive a camera as possible.

Advantageously, in step iii) of a method of the invention, the intensityof light output is monitored continuously. Preferably, the light outputis monitored continuously during at least a part of the amplificationreaction of step ii). More preferably, the light output is monitoredcontinuously during the whole of the amplification reaction of step ii).Step iii) also encompasses alternatively or additionally monitoring theintensity of the output of light continuously after the amplificationreaction of step ii) has finished.

A method according to the invention may be used to determine the amountof template nucleic acid present in a sample in a quantitative fashionand/or in a qualitative fashion.

Use in a quantitative fashion includes the use of a method of theinvention to determine the amount of template present in a sample priorto the nucleic acid amplification reaction of step ii) occurring. Italso includes the use of a method of the invention to determine theamount of template nucleic acid present in a sample as a result of theamplification reaction of step ii), which may be determined eitherduring or following the nucleic acid amplification reaction of step ii);i.e., the quantification of how much nucleic acid amplification product(“amplicon”) has been produced. This makes it possible to quantify theextent of the nucleic acid amplification reaction. When used in aquantitative fashion, the term ‘determine’ includes both an accuratedetermination of the amount of template nucleic acid present in thesample and an estimate of the amount of template nucleic acid present inthe sample.

It has surprisingly been found that in order to determine the amount oftemplate nucleic acid present in a sample in a quantitative fashion, thetiming of the change in intensity of light output is a proportionatefactor in addition to the intensity per se of the light output produced.For example, for a particular set of reaction conditions (e.g., aparticular template nucleic acid, a particular concentration ofcomponents for the amplification reaction and the bioluminescence assayand a particular temperature(s) for the amplification reaction), if ahigher concentration of template nucleic acid is present in the sampleat the beginning of the nucleic acid amplification reaction, the changesin intensity of light output will occur after a shorter period of timefollowing the start of the amplification reaction when compared to areaction in which a lower concentration of template nucleic acid ispresent in the sample. Thus, for a particular set of reactionconditions, it is possible to determine the amount of template nucleicacid that is present in the sample by monitoring the change in intensityof light output as a function of time. Preferably, a series of controlreactions are performed using different known concentrations of theparticular template nucleic acid under the particular set of reactionconditions and the results obtained from the sample under analysis by amethod of the invention are compared to the results obtained from thisseries of control reactions. A control can also be performed wherein theamount of template nucleic acid that has been produced during theamplification reaction at predetermined time points is assessed usinggel electrophoresis or another suitable quantitative method. This willenable the amount of template nucleic acid in the control sample at thepredetermined time point to be calculated and correlated with therespective points on the data set.

When used in a qualitative fashion, a method of the invention can beused to assess whether or not a nucleic acid amplification reaction hasproduced any amplification product and thereby determine whether anytemplate nucleic acid is present in the sample. In many applicationswhere the amplification conditions are already sufficiently optimised(e.g. rapid detection of nucleic acid (preferably DNA) associated withpathogens), the only information required to establish that the targetDNA sequence was present in a sample is the occurrence of theamplification reaction. Where template nucleic acid is present in thesample, this will result in amplicon being produced as a result of thenucleic acid amplification reaction of step ii). Consequently, this willresult in a change in the pattern of the intensity of light output as afunction of time when compared to a control reaction in which noamplification has taken place. Where no template nucleic acid is presentin the sample, no amplification reaction will take place in step ii) andthus no amplicon will be produced as a result. Consequently, the patternof change in intensity of light output as a function of time will besimilar if not the same as a control in which no amplification has takenplace. Thus, the expression ‘performing the nucleic acid amplificationreaction’ as used in step ii) includes both ‘performing the nucleic acidamplification reaction’ and also ‘creating the appropriate conditionsfor the amplification reaction to occur’, since in embodiments in whichthere is no template nucleic acid present in the sample, no nucleic acidamplification reaction will occur. Preferably, the presence or absenceof the expected light change is monitored with a predetermined period oftime following the start of the reaction.

As mentioned above, it has also been found that PPi can itself havedirect effects on luciferase at high concentrations. This applies toboth luciferases that use ATP as a substrate for the production of lightand also reversibly-inhibited luciferases. By carrying out a number ofcontrol experiments using different concentrations of a particularstarting template nucleic acid under a particular set of reactionconditions, the skilled person will be able to determine from the dataset the time at which PPi itself has a direct effect on the luciferase.These control results can then be used to extrapolate the amount oftemplate nucleic acid present in the sample.

For example, it has been found that PPi can itself inhibit luciferase athigh concentrations. The point at which the intensity of light outputbegins to rapidly decrease correlates with the point at which theluciferase has become inhibited by a particular concentration of PPi.This may correspond to the point at which the intensity of light outputis at a maximum, i.e., the point which marks the transition between thelight output increasing and the light output decreasing. Alternatively,it may represent the point at which the rate of decrease in intensity oflight output significantly increases, e.g. from a gradual decrease to arapid decrease. By carrying out a number of control experiments usingdifferent concentrations of template nucleic acid, the time at which theintensity of light output begins to rapidly decrease for each particularstarting template nucleic acid concentration under a particular set ofreaction conditions can be determined. These control results can then beused to extrapolate the amount of template nucleic acid present in thesample.

Alternatively, PPi may cause an increase in light emission from aluciferase inhibited by a substance other than PPi, as in thereversibly-inhibited luciferase embodiment mentioned above.

Thus whether or not PPi stimulates or inhibits the bioluminescence assaycatalysed by luciferase depends on a number of factors including theprecise type of luciferase used, the temperature of the reaction, theconcentration of PPi and the presence of other compounds that can affectluciferase activity. By carrying out a number of control experimentsusing different concentrations of a particular starting template nucleicacid under a particular set of reaction conditions, the skilled personwill be able to determine from the data set the time at which PPi itselfhas a direct effect on the luciferase and the nature of this effect.These control results can then be used to extrapolate the amount oftemplate nucleic acid present in the sample.

The data set of intensity of light output as a function of time can beinterpreted in a number of different ways in order to determine theamount of template nucleic acid present in the sample. Particular pointson the data set represent points in time at which specificconcentrations of PPi are present. These can then be correlated to theamount of template nucleic acid present in the sample. For example, oneor more of the following points on the data set are preferablymonitored: i) the time taken to reach the point at which the intensityof light output begins to increase; ii) the time taken to reach thepoint at which the rate of change of increase of intensity of lightoutput increases or decreases; iii) the time taken to reach the point atwhich the rate of change of intensity of light output changes from anincrease to a decrease (this is preferably the point of maximumintensity of light output or “peak” intensity of light output) or from adecrease to an increase; iv) the time taken to reach the point at whichthe rate of change of decrease in intensity of light output increases ordecreases, and/or v) the time taken to reach the point at which theintensity of light output reaches or crosses a predetermined level.

For determination of the amount of template nucleic acid present in thesample in a quantitative fashion, the points on the data set which aremonitored are preferably those points at which the rate of change inintensity of light output changes significantly. When interpreting thedata set, the points at which the rate of change in intensity of lightoutput changes significantly will be apparent to the skilled person.

Most preferably, a point at which the rate of change in intensity oflight output changes significantly will be a point which represents atransition between the intensity of light output increasing and theintensity of light output decreasing. A point which represents atransition between the intensity of light output decreasing and theintensity of light output increasing is also a point at which the rateof change in intensity of light output changes significantly. A pointwhich marks a transition between the intensity of light outputincreasing and decreasing or decreasing and increasing will preferablybe represented as an inflection point when the results are displayed ona graph of intensity of light output as a function of time. A point atwhich the intensity of light output changes from a constant intensity toan increase or decrease in intensity, or a point at which the intensityof light output changes from an increase or decrease in intensity to aconstant intensity also represents a point at which the rate of changein intensity of light output changes significantly.

Alternatively, a point at which the intensity of light output changessignificantly may be a point at which the rate of increase in intensityof light output or the rate of decrease in intensity of light outputsignificantly increases or decreases. Thus, the expression ‘a point atwhich the rate of change in intensity of light output changessignificantly’ preferably refers to a point at which the rate of changein intensity of light output at a predetermined time interval beforethat points differs by at least 30% from the rate of change in intensityof light output at the same predetermined time interval after thatpoint. More preferably, ‘a point at which the rate of change inintensity of light output changes significantly’ refers to a point atwhich the rate of change in intensity of light output at a predeterminedtime interval before that points differs by at least 50% from the rateof change in intensity of light output at the same predetermined timeinterval after that point. Even more preferably, ‘a point at which therate of change in intensity of light output changes significantly’refers to a point at which the rate of change in intensity of lightoutput at a predetermined time interval before that points differs by atleast 70% from the rate of change in intensity of light output at thesame predetermined time interval after that point. Alternatively, ‘apoint at which the rate of change in intensity of light output changessignificantly’ refers to a point at which the rate of change inintensity of light output at a predetermined time interval before thatpoint differs by at least 10%, 20%, 40%, 60% or 80% from the rate ofchange in light intensity at the same predetermined time interval afterthat point. The predetermined time interval is preferably 30 seconds butmay alternatively be 1 minute, 1 minute 30 seconds or more.Alternatively, the predetermined time interval may be less than 30seconds. The chosen predetermined time interval will depend upon thetime intervals at which the intensity of light output is monitored andwill depend upon the kinetics of the particular amplification reactionthat is being studied.

Thus for quantitative determination, one or more of the following pointson the data set are preferably monitored: i) the point at which theintensity of light output begins to increase; ii) the point at which therate of change of increase of intensity of light output significantlyincreases or decreases; iii) the point at which the rate of change ofintensity of light output changes from an increase to a decrease(preferably the point of maximum intensity of light output) or from adecrease to an increase and/or iv) the point at which the rate of changeof decrease in intensity of light output significantly increases ordecreases. The time at which the intensity of light output reaches orcrosses a predetermined level may also be monitored.

In embodiments in which a reversibly-inhibited luciferase is not used,the amount of nucleic acid present in the sample is preferablydetermined in a quantitative fashion by monitoring one or more of thefollowing points: i) the time taken to reach a point at which theintensity of light output to begin to increase; ii) the time taken toreach a point at which the rate of change of intensity of light outputto change from an increase to a decrease; iii) the time taken to reach apoint at which the rate of change of decrease in intensity of lightoutput significantly increases; iv) the time taken to reach a point atwhich the rate of change of decrease in intensity of light outputsignificantly decreases; and v) the time taken for the intensity oflight output to reach or cross a predetermined level.

In embodiments in which a reversibly-inhibited luciferase is used,whilst the luciferase is inhibited by the product of its reaction, theintensity of output of light decreases gradually. Then, once a certainamount of PPi is produced as a result of the amplification reaction, theluciferase becomes sensitive to PPi and thus uninhibited and theintensity of light output increases. The intensity of light output thengradually decreases until a certain amount of PPi has been produced atwhich point, the rate of change of decrease in intensity of light outputincreases and the intensity of light output then decreases to a levelthat is less than the intensity of light output of a control reaction inwhich no nucleic acid amplification has taken place. The rate of changeof decrease of intensity of light output then decreases. Thus, inembodiments in which a reversibly-inhibited luciferase is brought intoassociation with the reaction mixture in step i), the amount of templatenucleic acid present in the sample is preferably determined in aquantitative fashion by monitoring one or more of the following points:i) the time taken to reach a point at which the intensity of lightoutput changes from a gradual decrease to an increase (i.e. the point atwhich the intensity of light output begins to increase); ii) the timetaken to reach a point at which the rate of decrease in the intensity oflight output significantly increases (preferably from a gradual decreaseto a rapid decrease); and iii) the time taken to reach a point at whichthe rate of decrease in the intensity of light output significantlydecreases (preferably from a rapid decrease to a gradual decrease).

As mentioned above, the time it takes to reach a particular point for aparticular template nucleic acid depends upon the concentration oftemplate nucleic acid present in the sample at the beginning of theamplification reaction. Thus, step iv) of a method of the inventionpreferably further comprises comparing the intensity of light output tothe intensity of light output from a standard curve formed by theresults from a number of controls in which the samples comprise knownamounts of template nucleic acid in order to determine the amount oftemplate nucleic acid in the sample.

For determination of the amount of template nucleic acid present in thesample in a qualitative fashion, i.e., whether or not the templatenucleic acid is present in the sample, the point on the data set whichis monitored is preferably the point at which the intensity of lightoutput reaches or crosses a predetermined level.

In embodiments in which a reversibly-inhibited luciferase is not used,an increase in the intensity of light output will indicate the presenceof template nucleic acid in the sample. Preferably, the increase inintensity of light output is relative to a control reaction in which noamplification has taken place. For example, such a control reaction willpreferably be one in which no template nucleic acid is present or one inwhich no polymerase is present. Thus, in these embodiments, the amountof nucleic acid present in the sample may be determined in a qualitativefashion by monitoring whether the intensity of light output rises abovethat of a control in which no amplification has taken place. Morepreferably, in these embodiments, the amount of nucleic acid present inthe sample may be determined in a qualitative fashion by monitoringwhether the intensity of light output reaches or rises above apredetermined level. For example, the predetermined level could be setat 125% or 150% of the light output at the beginning of theamplification reaction at the point at which the rate of decrease inlight intensity is at a minimum. If the intensity of light outputreaches this predetermined level or increases beyond it, this willindicate the presence of template nucleic acid in the sample. However,if the intensity of light output does not reach this predeterminedlevel, this will indicate the absence of template nucleic acid in thesample.

The predetermined level may vary depending on one or more factorsincluding: the template nucleic acid used, the concentration of thecomponents used in the nucleic acid amplification reaction and thetemperature used for the nucleic acid amplification reaction. Bycarrying out control experiments in which template nucleic acid ispresent or template nucleic acid is not present, the skilled person willreadily be able to determine a suitable predetermined level.

Preferably, the presence of the increase in the intensity of lightoutput within a predetermined length of time following the start of theamplification reaction of step ii) indicates the presence of templatenucleic acid in the sample and the absence of the increase in theintensity of light output within the predetermined length of timefollowing the start of the amplification reaction of step ii) indicatesthe absence of template nucleic acid in the sample. For example, where amethod of the invention is used for genotyping, where a certain amountof test material would always contain a certain amount of targettemplate, then if the target template nucleic acid is present, one canconfidently state that if the intensity of light output has notincreased within a predetermined time, then the target is absent.

Preferably, the predetermined length of time will be a time which occursduring the amplification reaction of step ii). The less template nucleicacid that is present at the beginning of the reaction, the longer theamplification reaction of step ii) takes. By carrying out a number ofcontrol experiments for a particular template nucleic acid under aparticular set of reaction conditions in which template nucleic acid ispresent at varying concentrations or template nucleic acid is notpresent, the skilled person will readily be able to determine a suitablepredetermined time by which the increase must have or must not haveoccurred for that particular template nucleic acid under that particularset of reaction conditions. For example, the predetermined length oftime may be within 20, 25, 30, 35, 40, 45, 50 or more minutes from thestart of the nucleic acid amplification reaction.

Alternatively or additionally, in a method according to the invention, adecrease in the intensity of light output relative to a predeterminedlevel indicates the presence of template nucleic acid in the sample. Itis hypothesised that this decrease occurs when the luciferase becomesinhibited by PPi. For example, the predetermined level could be set at25%, 20%, 15%, 10% or 5% of the light output at the beginning of theamplification reaction at the point at which the rate of decrease inlight intensity is at a minimum. If the intensity of light outputdecreases to this predetermined level or decreases beyond it, this willindicate the presence of template nucleic acid in the sample. However,if the intensity of light output does not reach this predeterminedlevel, this will indicate the absence of template nucleic acid in thesample.

The predetermined level may vary depending on one or more factorsincluding: the template nucleic acid used, the concentration ofcomponents used in the nucleic acid amplification reaction and thetemperature of the nucleic acid amplification reaction. By carrying outcontrol experiments in which template nucleic acid is present ortemplate nucleic acid is not present, the skilled person will readily beable to determine a suitable predetermined level.

Step iv) of a method of the invention preferably further comprisescomparing the intensity of light output to the intensity of light outputfrom a control in which no amplification has taken place. For example,such a control may be one in which the same steps are carried out as ina method according to the invention except that either the templatenucleic acid and/or one of the other components needed for theamplification reaction (e.g. the polymerase) is/are omitted. This allowsthe decay of bioluminescence over time to be taken into account.

In a method according to the invention, although a control is preferablyrun simultaneously to the sample under analysis, it is not necessary forthis to be the case. For example, the control may be a control which hasbeen run previously and the data obtained therefrom could be used forcomparison with numerous other samples.

In a method according to the invention, a decrease in the intensity oflight output relative to a control reaction in which no amplificationhas taken place indicates the presence of template nucleic acid in thesample. This decrease relative to the control will occur subsequent tothe other changes in intensity of light output relative to the controlthat are described above. The finding that the intensity of light outputeventually decreases to a level that is less than a control reaction inwhich no amplification has taken place is surprising as the skilledperson would expect the intensity of light output to continue toincrease as more PPi is produced. It is hypothesised that the intensityof light output decreases to a level less than the control because theluciferase becomes inhibited by PPi. Although the monitoring of theintensity of light output to determine whether it is less than that of acontrol in which no amplification has taken place is preferably carriedout during the amplification reaction of step ii), it may alternativelybe carried out following the nucleic acid amplification reaction of stepii). Preferably, the intensity of light output decreases to a level thatis 30% or less of the intensity of light output of the control reaction.More preferably, the intensity of light output decreases to a level thatis 20% or less of the intensity of light output of the control reaction.Even more preferably, the intensity of light output decreases to a levelthat is 10% or less of the intensity of light output of the controlreaction. Alternatively, the intensity of light output may decrease to alevel that is 90% or less, 80% or less, 70% or less, 60% or less, 50% orless or 40% or less of the intensity of light output of the controlreaction.

Preferably, the presence of the decrease in the intensity of lightoutput relative to the predetermined level or to the control reactionwithin a predetermined length of time following the start of the nucleicacid amplification reaction indicates the presence of template nucleicacid in the sample and the absence of the decrease in the intensity oflight output relative to the predetermined level or to the controlreaction within the predetermined length of time following the start ofthe amplification reaction indicates the absence of template nucleicacid in the sample. The predetermined length of time is preferablywithin 20, 25, 30, 35, 40, 45, 50 or more minutes from the start of thenucleic acid amplification reaction. By carrying out control experimentsin which different concentrations of template nucleic acid are presentor template nucleic acid is not present, the skilled person will readilybe able to determine a suitable predetermined time by which the decreasemust have or must not have occurred.

The nucleic acid amplification reaction of step ii) is preferablycarried out within a temperature range in which the luciferase issufficiently active and stable to give sufficient and stable lightoutput over the duration of the amplification reaction. Further, theamplification reaction of step ii) is preferably one that can beperformed at a low enough temperature and that is rapid enough for theluciferase to remain stable during the amplification reaction. Thenucleic acid amplification reaction of step ii) of a method of theinvention may be carried out isothermally or may be a thermocyclingmethod. Preferably, the nucleic acid amplification reaction of step ii)of a method of the invention is carried out isothermally. Nucleic acidamplification reactions which are carried out isothermally are thosenucleic acid amplification reactions which do not rely on thermocyclingfor the amplification reaction to proceed.

Examples of nucleic acid amplification reactions which do not involve aRNA synthesis step and which are suitable for monitoring by a methodaccording to the invention include both isothermal methods and alsothermocycling methods such as PCR.

Isothermal methods which do not involve an RNA synthesis step proceedvia strand displacement. Such methods include: rolling circleamplification (see Fire, A. and Xu, S.-Q. (1995) ‘Rolling replication ofshort DNA circles’, Proc. Natl. Acad. Sci. USA, 92, 4641-4645), rollingcircle amplification technology (seehttp://www.molecularstaging.com/Pages/RCATdetails_.html; Amersham'sPhi29-based amplification Kit, product codes: 25-6400-10 and25-6400-50), isothermal ramification amplification (Zhang, W. et al.,‘Detection of Chlamydia trachomatis by isothermal ramificationamplification method: a feasibility study’, J. Clin. Microbiol., January2002, 128-132), restriction-endonuclease-dependent strand displacementamplification (Walker, G. T., ‘Isothermal in vitro amplification of DNAby a restriction enzyme/DNA polymerase system’, PNAS, (1992), 89,392-396), loop-mediated isothermal amplification (LAMP) (Notomi, T.,‘Loop-mediated isothermal amplification of DNA’, Nucl. Acids. Res.,2000, 28(12), e63, i-vii) and variants of these methods. Isothermalnucleic acid amplification techniques that do not involve anRNA-synthesis step and which proceed via strand-displacement mechanismsare also known as ‘isothermal PCR’ techniques. The finding that abioluminescence assay based on an ELIDA assay can be used to monitoramplification reactions that proceed via strand displacement issurprising given the number of background reactions that occur due tothe low temperature of the amplification reaction.

Alternatively, thermocycling methods which do not involve an RNAsynthesis may be used in a method of the invention provided that all thecomponents of the amplification reaction and the bioluminescence assayare stable at the temperatures through which the PCR cycles. Preferably,the thermocycling reaction is a low temperature thermocycling method inwhich primer extension is carried out in a cycling temperature rangethat does not exceed 75° C. and which preferably does not exceed 70° C.and which utilises a moderately thermostable DNA polymerase. Such amethod is LoTemp® PCR which uses a HiFi® DNA polymerase and is describedat www.hifidna.com/FQAall.htm. Alternatively, the thermocycling reactionis a low temperature thermocycling method which utilises the Klenowfragment of DNA polymerase I in the presence of proline (see NucleicAcid Research, (1999), 27(6), 1566-1568).

Examples of isothermal amplification reactions that involve an RNAsynthesis step and that can be monitored by a method of the inventioninclude transcription mediated amplification (TMA) or nucleic acidsequence based amplification (NASBA) (Guatelli, J. C. et al.,‘Isothermal, in vitro amplification of nucleic acids by a multienzymereaction modelled after retroviral replication’, PNAS, (1990), 87,1874-1878) and variants of these methods.

The nucleic acid amplification reaction of step ii) is carried outwithin a temperature range within which the components of theamplification reaction and the bioluminescence assay remain stable.Preferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range that does not exceed 75° C. Morepreferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range that does not exceed 70° C. Evenmore preferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range that does not exceed 65° C. Mostpreferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range that does not exceed 60° C.,i.e., a temperature range within which the Ultra-Glow thermostableluciferase from Promega is sufficiently active and stable to givesufficient and stable light output over the duration of theamplification reaction. Alternatively, the nucleic acid amplificationreaction of step ii) may be carried out within a temperature range thatdoes not exceed 55° C., 50° C., 45° C. or 40° C.

Preferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range that does not go below 20° C.More preferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range that does not go below 30° C.Even more preferably, the nucleic acid amplification reaction of stepii) is carried out within a temperature range that does not go below 40°C. Alternatively, the nucleic acid amplification reaction of step ii)may be carried out within a temperature range that does not go below 25°C., 35° C., 45° C., 50° C., 55° C. or 60° C.

Preferably, the nucleic acid amplification reaction of step ii) iscarried out within a temperature range of 30° C. to 75° C. Morepreferably, the nucleic acid amplification reaction of step ii) iscarried out within the temperature range of 30° C. to 65° C. Forexample, the nucleic acid amplification reaction of step ii) may becarried out within the temperature range of 45° C. to 65° C. or 35° C.to 40° C.

The nucleic acid amplification reaction of step ii) may be carried outat a constant temperature within the temperature ranges specified above.In a preferred embodiment, the nucleic acid amplification reaction iscarried out at 37° C. For example, by using a mutant firefly luciferaseenzyme that is stable at 37° C. (wild-type enzyme rapidly inactivates atthis temperature), one can monitor the generation of PPi during theisothermal nucleic acid amplification reaction using a standard ELIDAreaction. An example of a mutant firefly luciferase enzyme that isstable at 37° C. and which is suitable for use in a method of thepresent invention is described by Tisi, L. et al. (Tisi, L. C. et al.,(2002) ‘Development of a thermostable firefly luciferase’, AnalyticaChimica Acta, Vol. 457, 115-123).

Alternatively, the nucleic acid amplification reaction of step ii) maybe carried out at more than one temperature within the preferredtemperature range.

Where it is found that the luciferase that is used produces a loweroverall intensity of light output from the bioluminescence assay(whether amplification occurs or not) when the temperature at which thenucleic acid amplification reaction is performed is increased, it isadvantageous for the nucleic acid amplification reaction of step ii) tobe run at a lower temperature. This has the dual advantage that theintensity of light output is increased and that the amplificationreaction occurs more slowly. A slower amplification reaction isparticularly beneficial for quantitative analysis since the data pointswhich correspond to the various points at which there is a variation inthe rate of change of light intensity with time for samples havingdifferent amounts of the template nucleic acid occur over a greaterperiod of time than when the amplification reaction is monitored at thehigher temperature and are thus more easily monitored.

However, running the nucleic acid amplification reaction at a lowertemperature could potentially affect the specificity of theamplification reaction. For example, there could be a greater chance ofa false positive result as the temperature of the amplification reactionis reduced since the chance of primers annealing to sequences other thanthe desired target sequence increases as the temperature of the nucleicacid amplification reaction is reduced. Thus, the invention alsoprovides a method in which the nucleic acid amplification reaction ofstep ii) is started at a higher temperature and subsequently dropped toa lower temperature. Preferably, this higher and lower temperature arewithin a preferred temperature range as discussed above. This has theadvantage that the nucleic acid amplification reaction can be initiatedat a higher temperature where specificity is greater, then, beforeamplification enters a detectable exponential phase, the temperature canbe lowered to increase light intensity and slow the progress of theresults.

The relatively low temperature of the isothermal methods and the lowtemperature thermocycling method compared to methods which utiliseconventional thermocycling PCR in which the temperature is raised to 95°C. allows for smaller sample volumes to be analysed. In particular, inembodiments in which the temperature range does not exceed 55° C.,exquisitely small sample volumes can be analysed by a method of theinvention. For example, sample volumes of less than 10 μl and evensample volumes of less than 1 μl may be analysed by a method of theinvention. The high temperatures required in conventional PCR make verysmall sample volumes a technical challenge. The ability to analyse verysmall sample volumes also has the advantage of cutting reagent costs.

Thus, in a preferred embodiment, a method of the invention requires thatin the amplification reaction of step ii), the polymerase reaction isconducted isothermally and that the luciferase that is used is stable atthat temperature. This offers the following advantages:

-   i) the isothermal nucleic acid amplification reaction could be    monitored continuously in real-time;-   ii) the isothermal nucleic acid amplification reaction could be    monitored in a completely closed system without the need for further    reagent addition;-   iii) the relatively low temperature of the assay would allow    exquisitely small sample volumes to be analysed (the high    temperature of conventional PCR make very small samples volumes a    technical challenge so cutting reagent costs; and-   iv) a simple CCD camera could be employed to simultaneously monitor    thousands of isothermal PCR reactions.

It is a feature of the invention that PPi from nucleic acid synthesisduring nucleic acid amplification can be detected when the nucleic acidwhich has been synthesised would be undetectable by gel electrophoresis,resulting in increased sensitivity and reduced amplification time.Further, whilst the turbidity method of Mori et al (Mori, Y. et al.,Biochem. Biophys. Res. Comm., (2001) 289, 150-154) requires PPiconcentrations of ˜0.6 mM before significant turbidity is observed, byusing a pyrophosphate assay in which PPi is converted to ATP by ATPsulphurylase and by which the ATP produced is used by a luciferase toproduce light, PPi concentrations of less than 0.5 μM result in a linearrelationship between PPi concentration and bioluminescence (Nyren &Lundin, Analytical Biochemistry, 151(2), 405-409 (1985)). Thisrepresents an increase in sensitivity of a method of the invention fordetecting PPi of at least 1200 times over a turbidity assay. The methodsof the invention are also more sensitive than fluorescence-basedmethods.

A method according to the invention may be used in medical diagnosticapplications. At present, most diagnostic test centres need to send offtheir tests for analysis since conventional methods for analysingnucleic acid amplification reactions such as PCR require complicatedhardware and optics. The use of a method as described above will enabletest results to be analysed at point-of-care. For example, it could beused in sexual health clinics, for instance to see whether a pathogensuch as particular bacterium or virus is present in a sample. It mayalso be used to determine the amount of bacteria or virus present in asample, for example, to determine the extent of an infection.

A further application of a method according to the invention is fordetermining whether a particular nucleic acid sequence is present in anorganism's genetic code. For example, it could be used for determiningwhether the nucleic acid to which the template nucleic acid originateshas been genetically modified, for detection of DNA associated with aparticular non-genetically modified breed of plant or a geneticallymodified plant, for detection of DNA associated with pedigree breeds ofanimal or for medical or veterinary diagnostic applications such asgenetic testing or forensics.

A method according to the invention may be used to detect the presenceof an organism in a sample. As mentioned above, this organism may be apathogen. However, the method may also be used to detect anon-pathogenic organism.

A method of the invention may also be used in immuno-nucleic acidamplification technology (for example, see Sano, T. et al., (1992)Science, vol. 258, 120-122) (e.g., for identification of a particulartemplate nucleic acid linked to an antibody). The method is alsosuitable for use in situ where techniques such as fluorescence orabsorbance would be technically difficult to use. For example, a methodof the invention could be used on a metal surface. Thus a method of theinvention could be used, for example, to look for prions on a scalpelblade.

A kit for use in a method according to the invention preferablycomprises a nucleic acid polymerase, the substrates for the nucleic acidpolymerase, at least two primers, a thermostable luciferase, luciferinand optionally ATP sulphurylase and adenosine 5′ phosphosulphate. Morepreferably, the kit further comprises buffer reagents, such as a sourceof magnesium ions. Alternatively, a kit for use in a method according tothe invention may comprise only some of these components and/oradditional components. The sample and any other components that havebeen omitted from the kit may then be added to the kit during use.

For example, a kit for use in a method of the invention may comprisecontainers respectively containing:

-   a) a buffered mixture of nucleic acid polymerase, a source of Mg and    dNTPs; and-   b) a luciferase, luciferin and ATP sulphurylase.

Preferably, at least one of the components of the kit is lyophilised oris in another form which is suitable for storage in the kit. Morepreferably, all of the components of the kit are lyophilised or in oneor more other forms suitable for storage. Such other forms includecomponents to which stabilising factors have been added and/or arefrigerated or frozen mastermix that contains the components of thekit.

A preferred form of kit is a miniature “liquid” circuit. Preferably, akit for use in the present invention will be the size of a credit-cardfor ease of handling.

A kit for use in a method according to the invention can be used toanalyse one sample at a time or more than one sample at a time. Forexample, a kit for use in a method according to the invention may beused to monitor 2, 3, . . . , 50, . . . , 100, . . . 200 up to 1000 s ofsamples at a time.

In embodiments in which a method of the present invention is used tomonitor more than one sample at a time, the method may be for detectingthe presence of a template nucleic acid of the same sequence in eachsample or may be for detecting the presence of template nucleic acidshaving different sequences in different samples.

The results may be displayed on a test card that displays the resultsfrom one sample or more than one sample. Preferably, the test card isabout the size of a credit card for ease of handling.

The invention further provides a device for performing a method of theinvention and which incorporates the components that are present in akit according to the invention. For example, a device according to theinvention preferably incorporates a nucleic acid polymerase, thesubstrates for the nucleic acid polymerase, at least two primers, athermostable luciferase, luciferin and optionally ATP sulphurylase andadenosine 5′ phosphosulphate.

The invention will now be described further by way of example only withreference to the following figures in which:

FIG. 1 shows a set-up used to follow a LAMP reaction;

FIG. 2 shows the output from LAMP in the presence of target DNA and in acontrol without Bst DNA Polymerase;

FIG. 3 shows the results from duplicate LAMP samples and duplicatecontrols;

FIG. 4 shows the results from samples prepared as in FIGS. 2 & 3 butshowing differences in absolute light intensity;

FIG. 5 shows the light emission profiles for LAMP using differentamounts of target template (duplicates) at 55° C.;

FIG. 6 shows the time to peak light emission;

FIG. 7 shows a plot of the raw output from a LAMP reaction intriplicate;

FIG. 8 shows plots of the 1^(st) derivative of the curves shown in FIG.7;

FIG. 9 shows a comparison of controls to samples;

FIG. 10 shows a LAMP reaction where the temperature is decreased from55° C. to 50° C. after 10 minutes;

FIG. 11 shows a plot of the light intensity against time for ATPSulphurylase-free LAMP with different amounts of starting template; and

FIG. 12 shows a differential plot (control subtracted) of the normalizedlight-outputs for the ATP Sulphurylase-free LAMP reactions of samplescontaining different amounts of target template.

EXAMPLES Example 1 Demonstration of a Method of the Present Invention

The isothermal nucleic acid amplification reaction known asLoop-Mediated Amplification (LAMP) was selected to exemplify thepotential for using a simple bioluminescent assay to follow nucleic acidamplification in real-time.

The present, most rapid manifestation of the LAMP method uses sixprimers. This manifestation has been demonstrated to detect 10⁵ copiesof target DNA in just 15 minutes (Nagamine et al. 2002 Molecular andCellular Probes, 16, p223-229). LAMP reactions normally run at 60-65° C.and require at least 4 mM of Magnesium ions.

In order to demonstrate a real-time bioluminescent output from a LAMPreaction in particular, it was necessary to find means to lower thetemperature at which the LAMP reaction runs. This is due to the factthat at temperatures as high as 65° C. even the most thermostable beetleluciferase known to date (the Ultra-Glow thermostable luciferase fromPromega) is not sufficiently active and/or stable to give sufficient andstable light output over the duration of a LAMP amplification (around 45minutes or longer may be required to confirm that a sample does notcontain any of a particular target DNA molecule).

It was recognized that lowering the concentrations of Magnesium ionsfrom 4 mM to 2 mM allowed LAMP reactions to run successfully at lowertemperatures. Further, high concentrations of Betaine can reduce theability of LAMP reactions to reproducibly run successfully at lowertemperatures. Finally, appropriate stabilizing agents that did notinterfere with the LAMP reaction were selected and included in theformulations. As a result, it was possible to formulate conditions wherea bioluminescence assay could occur simultaneously with a LAMP reactionover the full period of the amplification.

Starting Materials

1) Reaction Mixture (Less Bst-DNA Polymerase or Target DNA)

Quantity Reagent Supplier 20 mM Tris-acetate Sigma 10 mM KCl ″ 10 mMAmmonium Sulphate ″ 2 mM Magnesium Sulphate ″ 0.10% V/V Triton X-100 ″0.5% W/V BSA ″ 5% W/V Trehalose ″ 0.4 mg/ml Polyvinylpyrrolidone ″ 9 mMDithiothreitol Melford 100 μg/ml D-luciferin (Potassium Salt) Europa 54ng/ml Ultra-Glow rLuciferase Promega 100 μM Adenosine 5′ phosphosulphateSigma 0.5 U/ml ATP Sulphurylase ″ 250 μM Each of the four dNTPs AmershamBiosci. 0.8 μM Lamp B1cB2 primer PNAC Cambridge UK 0.8 μM Lamp F1F2cprimer ″ 0.4 μM Lamp Loop B primer ″ 0.4 μM Lamp Loop F primer ″ 0.2 μMLamp B3 primer ″ 0.2 μM Lamp F3c primer ″ pH 8.8 @ 25° C. (see below forprimer sequences)2) DNA polymerase

-   8 U/μl Bst DNA Polymerase New England Biolabs    3) Template DNA (SEQ ID NO:1)

catgaattctgtcaagtctacgataacttagcgcttaggatgtcagatacttatgatgataagctgatagactatcttgcctggaagcttacttcataatggatgacgtatgccatgatagataccattgtctagacataagactttcaatctgcatagtcatgatcgatccatgctcgagtccaagctagtcatagcttatcatcaactgaatctagtaagtcattgaattctagPrimer sequences (SEQ ID NOS:2-7):

Lamp B1cB2: tat cat ggc ata cgt cat cca ttt tta taa gct gat aga cta tcttgc Lamp F1F2c: tca atc tgc ata gtc atg atc gtt ttt tga tga taa gct atgact agc Lamp Loop B: tat gaa gta agc ttc cag Lamp Loop F: atc cat gctcga gtc caa Lamp B3 primer atg tca gat act tat gat g Lamp F3c primer aatgac tta cta gat tca gMethod

To a 200 μl PCR tube, 18.6 μl of the reaction mixture was added followedby 1 μl of 0.4 ng/μl Template DNA and 0.4 μl of Bst DNA polymerase. As acontrol in a further 200 μl PCR tube, 18.6 μl of the reaction mixtureand 0.4 ng/μl of template DNA were added but no Bst DNA polymerase.

The samples were placed on a heating block held at 50° C. that had beenplaced inside a Syngene GeneGenius light cabinet (www.syngene.co.uk).Using the Syngene Genesnap software (www.syngene.co.uk), light emissionfrom the samples was recorded (through the closed lids of the PCR tubes)in a series of pictures taken with a CCD camera within the Syngene lightcabinet (FIG. 1). Each picture represented the integrated light emissionfrom the sample over a period of 1 minute.

A total of 40 frames were recorded, hence the LAMP reaction was observedfor 40 minutes in total.

Results

Using Syngene software, the light output from each of the samples wasquantified as a function of time. The results obtained are shown in FIG.2.

Using agarose gel electrophoresis it was confirmed that the ‘sample’(with the template nucleic acid) had indeed amplified significantamounts of DNA while the control had synthesized none.

A number of features were noted about the light emission that resultedin the case of the amplification:

-   -   ii) Initially the rate of light decrease for the sample and the        control were similar;    -   ii) After a period, the light intensity from the sample started        to increase, whilst the control continued to decrease gradually;    -   iii) The rate of increase in light emission from the sample        increased, reached a maximum, then decreased until a point was        reached where the greatest magnitude of light emission during        the LAMP reaction was recorded;    -   iv) Following this maximum in light emission from the sample, a        decrease in light emission was observed;    -   v) The rate of decrease in light emission increased following        the maximal light emission and the magnitude of the light        emission became less than that of the control;    -   vi) The rate of decrease in light emission decreased and        eventually became similar to that of the control;    -   vii) At the end of the 40 minutes, the magnitude of light        emission from the sample was considerably less than the control        even though, in this case, the starting light intensity of the        sample was slightly higher (which is related to the fact that        the light emission from the samples was not processed in any way        to take account of the relative position of the samples relative        to the camera).

It is hypothesised that the decrease in light intensity following thepeak in light intensity is as a result of luciferase becoming inhibitedby pyrophosphate. As such, in the LAMP reaction, the peak in lightintensity represents a point in time when a specific amount ofpyrophosphate has accumulated. Therefore, the peak in light intensityrepresents a point in time when a specific amount of DNA has beensynthesized.

Example 2 Reproducibility of the Method of the Invention Using a LampAmplification Reaction

The same procedure was carried out as in example 1 except that multiplesamples were used to assess the reproducibility of results obtained inthe LAMP reaction.

Starting Materials and Methods

As for example 1 except the sample and control were performed induplicate or triplicate and the temperature of the reaction was raisedto 55° C.

Results

The results are shown in FIG. 3. The same progress of the sample curveas in Example 1 is seen in this case.

In this example both the rate of change of light emission and the timeto maximal light emission are extremely similar for both of the samples.Again, generation of amplified DNA in the samples was confirmed byagarose gel electrophoresis. For the controls, whilst the rate of changeof light emission for both cases are similar, there is a smalldifference in absolute value. Again, this is thought to be because ofthe effects associated with light capture by the system used rather thanany biochemical aspect. Nonetheless, even without data manipulation,clear-cut results can be obtained.

In some cases, the absolute light intensity observed within e.g.triplicate samples could vary due to light capture effects. Nonethelessthe rate of light change and the time to maximal light emission issimilar (FIG. 4).

Example 3 Use of a Method of the Invention in a Quantitative Fashion

Starting Materials and Methods

The same procedure outlined in example 1 was repeated but with differentamounts of target DNA in the samples. Duplicate samples were set upcontaining a total of either 0.4 ng, 40 pg, 4 pg or 0.4 pg of templatenucleic acid. The temperature of the LAMP reaction was 55° C.

Results

The resulting light emission profiles for each of the samples is shownin FIG. 5. The results obtained in FIG. 5 demonstrate a key property ofmethods of the invention. Whilst there is not a convincing correlationbetween the amount of target template and absolute light emission, thereis a clear relationship between the time to peak light emission or thetime to changes in the rate of change of light emission.

A plot of time to peak light emission against amount of target DNAdemonstrates that the correlation is quantitative (see FIG. 6 a in whichthe time to peak light emission has a linear correlation with the log 10of the concentration of DNA target Template in the sample). In FIG. 6 b,the time to produce 25% of the final total amount of amplicon is plottedwith the time to peak light emission and it can be seen that the twoparameters correlate. Thus, comparing the times to peak light emissionagainst results obtained with agarose gel electrophoresis demonstratesthat the time to peak light emission reflects the accumulation ofamplicon and thus the amount of template nucleic acid present in thesample

Example 4 Data Manipulation of Results from a Method of the Invention inwhich the Nucleic Acid Amplification Reaction is a LAMP Reaction

The same procedure as in example 1 was carried out again but usingmultiple samples to assess the reproducibility of results obtained inthe LAMP reaction after some simple data manipulation of the raw datahad been performed. Specifically, the 1^(st) derivative of the outputswere plotted.

Starting Materials and Methods

These were as for Example 1 except that the sample and control wereperformed in triplicate, the temperature was 50° C. and a total of 1 ngof template was used in each sample.

FIG. 7 shows a plot of the raw data from a method of the invention onthese samples. By plotting the rate of change in light emission overtime as opposed to the light intensity over time (i.e. plotting the1^(st) derivative), inflection points are highlighted. In particular,regions of the curves shown in FIG. 7 that go through minima or maximaintersect the Y axis at zero when the 1^(st) derivative of the curve isplotted (FIG. 7). While the magnitude of the intensities showsconsiderable variance, inflection points within sets of the curves aresimilar.

The curves in FIG. 8 for the samples where a LAMP amplification reactionhas occurred show two points crossing the Y-axis. The first representsthe first inflection point of FIG. 7 and the second represents the pointof maximum light intensity. The minima and maxima seen in FIG. 8highlight time-points associated with maximal rates of change in lightemission. All four data points (the two Y-axis intersections and theminima and maxima) show good superposition between the triplicatesamples. Note that the first Y-axis intersection occurs almost tenminutes before the second.

FIG. 9 shows an expanded view of the curves of FIG. 8 and highlights howplotting the 1^(st) derivative differentiates the sample from thecontrol.

Thus due to the inherent information content of the raw data from amethod of the invention using a LAMP reaction, even very simple datamanipulation, such as taking the 1^(st) derivative, not only allowsclear points on the resulting curves to be identified (Y-axisintersections and maxima and minima) but also makes the results lesssensitive to the magnitude of the light signals (e.g. compare thesuperposition of the curves in FIGS. 7 and 8—in FIG. 8 the superpositionis more similar).

Example 5 Changing Temperature of the Amplification Reaction DuringAmplification

The LAMP method can be made to work over the temperature range ofapproximately 45° C. to 65° C. However, the higher the temperature atwhich the LAMP reaction is run, the lower the overall light intensityfrom a method of the invention (whether amplification occurs or not).This is due to the particular thermostable luciferase used (theUltra-Glow luciferase from Promega) apparently catalysing the lightreaction at a lower rate at higher temperatures. Thus the rate of lightemission observed for the Ultra-Glow luciferase at 55° C. isconsiderably less than that observed at 50° C. However, on cooling frome.g. 55° C. to 50° C., one observes an increase in the rate of lightemission catalyzed by the Ultra-Glow luciferase, hence the effect isclearly reversible. The reversibility implies that the observed decreasein light emission at high temperature is not solely the result of theluciferase denaturing.

Running a LAMP reaction at a lower temperature hence increases the lightemission from the assay. Further, running LAMP at lower temperatures canslow the reaction itself. This may be beneficial in certaincircumstances. For example, when using a method of the inventionquantitatively, there may be benefits in slowing the amplification sothat the times taken to, e.g. reach peak light emission for samples withdifferent amounts of target template, are more greatly separated in timethan when the LAMP reaction is run at a higher temperature.

However, running LAMP reactions at low temperatures could potentiallyaffect the specificity of the process, that is, there could be a greaterchance of a false-positive result as the temperature of the LAMP isreduced. In other words, the chances of primers annealing to sequencesother than the desired target sequence, increases as the temperature ofthe LAMP reaction is reduced.

A possible compromise to take advantage of benefits of running the LAMPreaction at low temperatures and yet maintaining the maximal specificityis to change temperature during the LAMP reaction. Specifically, theLAMP reaction can be initiated at a higher temperature where specificityis greater, then, before amplification enters a detectable exponentialphase, the temperature can be lowered to increase light intensity andslow the progress of results.

Starting Materials and Methods

As for Example 1 except that a variety of samples were tested withdifferent amounts of target template as in Example 3 (over the range0.02 pg/μl to 20 pg/μl, i.e., 0.4 pg total sample to 0.4 ng totalsample). The LAMP reaction was initiated at 55° C. then the temperaturelowered to 50° C. after 10 minutes.

Results

The raw data obtained from the temperature change data is shown in FIG.10. The data shown in FIG. 10 show that the temperature change methoddoes indeed result in an increase in the intensity of light emission ondropping the temperature. Further, the LAMP remains quantitative, inthat the time to peak light emission remains a function of the startingamount of template DNA. Comparing FIG. 10 to FIG. 5, where equivalentamounts of target template are tested with LAMP but at a singletemperature of 55° C., it can be seen that the time difference betweenthe sample with the most template (0.4 ng total/20 pg/ul) and leasttemplate (0.4 pg total/0.02 pg/ul) is approximately 8 minutes, whereasin the temperature change method shown in FIG. 10, the time differenceis approximately 14 minutes.

In fact, temperatures higher than 55° C. may initially be used, since,though the Ultra-Glow luciferase begins to become unstable above 60° C.,it can tolerate being at higher temperatures for short periods. Thisapproach therefore increases the temperature ranges available.

Further, this approach may enable less stable luciferases to be employedwhere the amplification reaction does not require long periods attemperatures that can irreversibly inactivate luciferase.

Finally, whilst clearly false-positives can still occur using thetemperature change method, they should be less common due to theincreased stringency of the higher temperatures at the initial key phaseof amplification (i.e. just prior to exponential phase).

Example 6 Reversibly-Inhibited Luciferase-Based Method

As discussed above, pyrophosphate has direct effects on luciferase.Firstly, under certain circumstances, pyrophosphate can relieve theinhibition luciferase undergoes in the presence of certain inhibitorsincluding oxyluciferin, the product of the light reaction. Secondly,pyrophosphate can itself inhibit luciferase at high concentrations.Whether or not pyrophosphate stimulates or inhibits the light emittingreaction catalyzed by luciferase depends on a number of factorsincluding the precise type of luciferase, temperature, concentration ofpyrophosphate, presence of other compounds that can affect luciferaseactivity. This example shows how the inhibitory effect of pyrophosphatecan be used to follow a LAMP reaction. A vital aspect of this approachis that the presence of ATP in the sample can be tolerated: in methodsin which the bioluminescence assay relies on the detection of ATP by theluciferase for the production of light, significant amounts ofendogenous ATP in the sample would severely compromise the use of theassay.

The fact that the ATP Sulphurylase-free method tolerates ATP (in fact itworks best in the presence of ATP) means that it can potentially be usedto assay for pyrophosphate in amplification reactions that include anRNA synthesis step such as Transcription Mediated Amplification (TMA).

By carrying out control experiments under a particular set of reactionconditions, the skilled person will be able to determine the particularratio of luciferase to luciferin to ATP to pyrophosphate(luciferase:luciferin:ATP:PPi) that is required for use in a method ofthe invention.

Starting Materials and Methods

1) ATP Sulphurylase-Free Reaction Mixture (Less Bst-DNA Polymerase orTarget DNA)

Quantity Reagent Supplier 20 mM Tris-acetate Sigma 10 mM KCl ″ 10 mMAmmonium Sulphate ″ 2 mM Magnesium Sulphate ″ 0.10% V/V Triton X-100 ″0.5% W/V BSA ″ 5% W/V Trehalose ″ 0.4 mg/ml Polyvinylpyrrolidone ″ 9 mMDithiothreitol Melford 1 μg/ml D-luciferin (Potassium Salt) Europa 36ng/ml Ultra-Glow rLuciferase Promega 1 mM Adenosine triphosphate (ATP)Sigma 250 μM Each of the four dNTPs Amersham Biosci. 0.8 μM Lamp B1cB2primer PNAC Cambridge UK 0.8 μM Lamp F1F2c primer ″ 0.4 μM Lamp Loop Bprimer ″ 0.4 μM Lamp Loop F primer ″ 0.2 μM Lamp B3 primer ″ 0.2 μM LampF3c primer ″ pH 8.8 @ 25° C. (see below for primer sequences)Method

Samples were prepared as in Example 1 except using the reaction mixturedescribed above. A range of target template DNA concentrations weretested, from 1 pg to 1 ng. The ATP sulphurylase-free LAMP was run at 55°C. A Bst DNA polymerase-free sample was used as the control.

Results

The raw data resulting from the ATP sulphurylase-free LAMP are shown inFIG. 11. The raw data shown in FIG. 11 shows that the samples containingtemplate nucleic acid show a characteristic sudden decrease in lightintensity over time, not seen in the control. This decrease is believedto be the result of pyrophosphate produced by the LAMP reactioninhibiting the luciferase. As such the sudden decrease is a marker fornucleic acid amplification. That DNA synthesis was actually occurringwas confirmed by agarose gel electrophoresis of the samples.

Data manipulation of the raw data enabled further interpretation of theresults. Firstly, the data were normalized to their starting lightintensities, then the values obtained from the control lackingpolymerase were subtracted from that of the template nucleicacid-containing samples (FIG. 12).

Examination of FIG. 12 indicates that the ATP sulphurylase-free LAMP isalso quantitative, as the time taken to reach points where the rate ofchange of light intensity significantly changes, appears to beproportional to the concentration of target template in the samples.

Since 1 mM ATP is present during the ATP sulphurylase-free LAMP, thesame approach can therefore be taken to follow RNA-based amplificationmethods.

It will be appreciated that the invention has been described above byway of example only and that further modifications in detail may be madewhich remain within the scope of the invention as defined by the claims.

1. A method for determining the amount of template nucleic acid presentin a sample comprising: i) bringing into association with the sample allthe components necessary for nucleic acid amplification, and all thecomponents necessary for a bioluminescence assay for nucleic acidamplification including: a) a nucleic acid polymerase, b) the substratesfor the nucleic acid polymerase, c) at least two primers, d) athermostable luciferase, e) luciferin, f) ATP sulphurylase, and g)adenosine 5′ phosphosulphate; and subsequently: ii) performing a nucleicacid amplification reaction of the template nucleic acid involving morethan one cycle of amplification; iii) monitoring the intensity of lightoutput from the bioluminescence assay; and iv) determining the amount oftemplate nucleic acid present in the sample.
 2. A method according toclaim 1, wherein at least ii) and iii) are carried out in a sealedvessel.
 3. A method according to claim 1, wherein in iii) the intensityof light output is monitored during the nucleic acid amplificationreaction.
 4. A method according to claim 1, wherein iii) furtherincludes producing a data set of intensity of light output as a functionof time.
 5. A method according to claim 4, wherein the amount oftemplate nucleic acid present is determined by measuring from the dataset the time taken to reach a point at which the rate of change ofintensity of light output changes significantly.
 6. A method accordingto claim 1, wherein the amount of template nucleic acid present in thesample before the nucleic acid amplification reaction of ii) isdetermined.
 7. A method according to claim 1, wherein the amount oftemplate nucleic acid present in the sample after the nucleic acidamplification reaction of ii) is determined.
 8. A method according toclaim 5, wherein the amount of template nucleic acid present isdetermined by measuring from the data set the time taken to reach apoint at which the intensity of light output begins to increase.
 9. Amethod according to claim 5, wherein the amount of template nucleic acidpresent is determined by measuring from the data set the time taken toreach a point at which the intensity of light output is at a maximum.10. A method according to claim 5, wherein the amount of templatenucleic acid present is determined by measuring from the data set thetime taken to reach a point at which the rate of decrease of intensityof light output increases.
 11. A method according to claim 5, whereinthe amount of template nucleic acid present is determined by measuringfrom the data set the time taken to reach a point at which the rate ofdecrease of intensity of light output decreases.
 12. A method accordingto claim 5, wherein the amount of template nucleic acid present isdetermined by measuring from the data set the time taken to reach apoint at which the intensity of light output reaches or crosses apredetermined level.
 13. A method according to claim 8, wherein thethermostable luciferase that is brought into association with the samplein i) is a reversibly-inhibited luciferase.
 14. A method according toclaim 1, wherein iv) further comprises comparing the intensity of lightoutput to the intensity of light output from a control in which thesample comprises a known amount of template nucleic acid.
 15. A methodaccording to claim 1 for determining whether the template nucleic acidis present in the sample, wherein whether the template nucleic acid ispresent in the sample is determined by measuring from the data setwhether the intensity of light output reaches or crosses a predeterminedlevel.
 16. A method according to claim 15, wherein an increase in theintensity of light output relative to the predetermined level indicatesthe presence of template nucleic acid in the sample.
 17. A methodaccording to claim 15, wherein a decrease in the intensity of lightoutput relative to the predetermined level indicates the presence oftemplate nucleic acid in the sample.
 18. A method according to claim 15,wherein whether the template nucleic acid is present in the sample isdetermined by measuring from the data set whether the intensity of lightoutput reaches or crosses the predetermined level within a predeterminedlength of time following the start of the amplification reaction of ii).19. A method according to claim 1, wherein iv) further comprisescomparing the intensity of light output to the intensity of light outputfrom a control in which no amplification has taken place.
 20. A methodaccording to claim 19, wherein a decrease in the intensity of lightoutput relative to a control reaction in which no amplification hastaken place indicates the presence of template nucleic acid in thesample.
 21. A method according to claim 1, wherein the nucleic acidamplification reaction of ii) is a low temperature thermocyclingamplification method in which the cycling temperature range does notexceed 75° C.
 22. A method according to claim 1, wherein the nucleicacid amplification reaction of ii) is carried out isothermally.
 23. Amethod according to claim 22, wherein the nucleic acid amplificationreaction of ii) is carried out within a temperature range that does notexceed 75° C.
 24. A method according to claim 22, wherein the nucleicacid amplification reaction of ii) is carried out at a constanttemperature at which the components of the amplification reaction andthe bioluminescence assay are stable.
 25. A method according to claim22, wherein the nucleic acid amplification reaction of ii) is carriedout at more than one temperature within the temperature range in whichthe components of the amplification reaction and the bioluminescenceassay are stable.
 26. A method according to claim 25, wherein thenucleic acid amplification reaction of ii) is started at a highertemperature and subsequently dropped to a lower temperature.
 27. Amethod according to claim 1 further comprising determining a medicaldiagnosis.
 28. A method according to claim 1 further comprisingdetermining whether a pathogen is present in a sample.
 29. A methodaccording to claim 1 further comprising determining whether a particularnucleic acid sequence is present in an organism's genetic code.
 30. Amethod according to claim 29 further comprising determining whether thenucleic acid to which the template nucleic acid originates has beengenetically modified.
 31. A method according to claim 1 furthercomprising determining whether an organism is present in a sample.
 32. Amethod according to claim 1, wherein the template nucleic acid is linkedto an antibody.
 33. A method for determining the amount of templatenucleic acid present in a sample comprising: i) bringing intoassociation with the sample all the components necessary for nucleicacid amplification, and all the components necessary for abioluminescence assay for nucleic acid amplification including: a) anucleic acid polymerase, b) the substrates for the nucleic acidpolymerase, c) at least two primers, d) a thermostable luciferase, ande) luciferin; and subsequently: ii) performing a nucleic acidamplification reaction of the template nucleic acid involving more thanone cycle of amplification; iii) monitoring the intensity of lightoutput from the bioluminescence assay; and iv) determining the amount oftemplate nucleic acid present in the sample.